Bulk RNA-seq analysis of developing ocular anterior segments from wildtype and Col4a1 mutant mice
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE202801 | GEO | 2022/05/18
REPOSITORIES: GEO
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Project description:Bulk RNA-seq analysis of developing ocular anterior segments from wildtype and Col4a1 mutant mice
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Project description:FOXE3 is a lens specific transcription factor that has been associated with anterior segment ocular dysgenesis. To determine the transcriptional target(s) of FOXE3 that are indispensable for the anterior segment development, we examined the transcriptome and the proteome of cells expressing truncated FOXE3 responsible for Peters anomaly identified through linkage-coupled next-generation whole exome sequencing. We found that DNAJB1, an autophagy-associated protein, was the only candidate exhibiting differential expression in both screens. We confirmed the candidacy of DNAJB1 through chromatin immunoprecipitation and luciferase assays while knockdown of DNAJB1 in human lens epithelial cells resulted in mitotic arrest. Subsequently, we targeted dnajb1a in zebrafish through injection of a splice-blocking morpholino. The dnajb1a morphants exhibited underdeveloped cataractous lenses with persistent apoptotic nuclei. In conclusion, here we report DNAJB1 as a transcriptional target of FOXE3 in a novel pathway that is crucial for the development of the anterior segment of the eye.
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Project description:Anterior segment dysgenesis (ASD), resulting in vision impairment, stems from maldevelopment of anterior segment (AS) tissues. Incidence of ASD has been linked to malfunction of periocular mesenchyme cells (POM). POM cells specify into anterior segment mesenchyme (ASM) cells which colonize and produce AS tissues. In this study we uncover ASM developmental trajectories associated with formation of the AS
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Project description:The anterior segment of the eye consists of the cornea, iris, ciliary body, crystalline lens and aqueous humor outflow pathways. Together, these tissues are essential for the proper functioning of the eye. Disorders of vision have been ascribed to defects in all of them; some, including glaucoma and cataract, are among the most prevalent causes of blindness. To characterize the cell types that comprise these tissues, we generated an anterior segment cell atlas of the human eye using high throughput single-nucleus RNA sequencing (snRNAseq). We profiled 191,992 single nuclei from non-diseased anterior segment tissues from 6 human donors, identifying >60 cell types. Many are discrete, whereas others in lens and cornea form continua, corresponding to known developmental transitions that persist in adulthood. Having profiled each tissue separately, we performed an integrated analysis of the entire anterior segment revealing that some cell types are unique to single structure whereas others are shared across tissues. This integrated cell atlas was then used to investigate cell type-specific expression patterns of more than >900 human ocular disease genes identified either through Mendelian inheritance patterns or genome-wide association studies (GWAS).
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Project description:FOXE3 is a lens specific transcription factor that has been associated with anterior segment ocular dysgenesis. To determine the transcriptional target(s) of FOXE3 that are indispensable for the anterior segment development, we examined the transcriptome and the proteome of cells expressing truncated FOXE3 responsible for Peters anomaly identified through linkage-coupled next-generation whole exome sequencing. We found that DNAJB1, an autophagy-associated protein, was the only candidate exhibiting differential expression in both screens. We confirmed the candidacy of DNAJB1 through chromatin immunoprecipitation and luciferase assays while knockdown of DNAJB1 in human lens epithelial cells resulted in mitotic arrest. Subsequently, we targeted dnajb1a in zebrafish through injection of a splice-blocking morpholino. The dnajb1a morphants exhibited underdeveloped cataractous lenses with persistent apoptotic nuclei. In conclusion, we have identified DNAJB1 as a transcriptional target of FOXE3 in a novel pathway that is crucial for development of the anterior segment of the eye.
2016-03-24 | GSE76389 | GEO
Project description:Mutations in Peroxidasin (PXDN) cause severe inherited eye disorders in humans, such as congenital cataract, corneal opacity, and developmental glaucoma. The role of peroxidasin during eye development is poorly understood. Here we describe the first Pxdn mouse mutant which was induced by ENU (N-ethyl-N-nitrosourea) and led to a recessive phenotype. Sequence analysis of cDNA revealed a T3816A mutation resulting in a premature stop codon (Cys1272X) in the peroxidase domain. This mutation causes severe anterior segment dysgenesis and microphthalmia resembling the manifestations in patients with PXDN mutations. The proliferation and differentiation of the lens is disrupted in association with aberrant expression of transcription factor genes (Pax6 and Foxe3) in mutant eyes. Additionally, Pxdn is involved in the consolidation of the basement membrane and lens epithelium adhesion in the ocular lens. Lens material including γ-crystallin is extruded into the anterior and posterior chamber due to local loss of structural integrity of the lens capsule as a secondary damage to the anterior segment development leading to congenital ocular inflammation. Moreover, Pxdn mutants exhibited an early-onset glaucoma and progressive retinal dysgenesis. Transcriptome profiling revealed that peroxidasin affects the transcription of developmental and eye diseases-related genes at early eye development. These findings suggest that peroxidasin is necessary for cell proliferation and differentiation and for basement membrane consolidation during eye development. Our studies provide pathogenic mechanisms of PXDN mutation-induced congenital eye diseases.
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Project description:Although the visual system extends through the brain, most vision loss originates from defects in the eye. Its central element is the neural retina, which senses light, processes visual signals, and transmits them to the rest of the brain through the optic nerve (ON). Surrounding the retina are numerous other structures, conventionally divided into anterior and posterior segments. Here we used high-throughput single nucleus RNA sequencing (snRNA-seq) to classify and characterize cells in the extraretinal components of the posterior segment: ON, optic nerve head (ONH), peripheral sclera, peripapillary sclera (PPS), choroid, and retinal pigment epithelium (RPE). Defects in each of these tissues are associated with blinding diseases – for example, glaucoma (ONH and PPS), optic neuritis (ON), retinitis pigmentosa (RPE), and age-related macular degeneration (RPE and choroid). From ~151,000 single nuclei, we identified 37 transcriptomically distinct cell types, including multiple types of astrocytes, oligodendrocytes, fibroblasts, and vascular endothelial cells. Our analyses revealed a differential distribution of many cell types among distinct structures. Together with our previous analyses of the anterior segment and retina, the new data complete a “Version 1” cell atlas of the human eye. We used this atlas to map the expression of >180 genes associated with the risk of developing glaucoma, which is known to involve ocular tissues in both anterior and posterior segments as well as neural retina. Similar methods can be used to investigate numerous additional ocular diseases, many of which are currently untreatable.
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Project description:Zebrafish with loss-of-function alx1 mutations develop with craniofacial and ocular defects of variable penetrance, likely due to compensatory upregulation in expression of a paralogous gene, alx3. We show that zebrafish alx1; alx3 mutants develop with highly penetrant cranial and ocular defects that resemble human ALX1-linked FND. alx1 and alx3 are expressed in anterior cranial neural crest (aCNC), which gives rise to the anterior neurocranium (ANC), anterior segment structures of the eye and vascular pericytes. Consistent with a functional requirement for alx genes in aCNC, alx1; alx3 mutants develop with nearly absent ANC and grossly aberrant hyaloid vasculature and ocular anterior segment, but normal retina. In vivo lineage labeling identified a requirement for alx1 and alx3 during aCNC migration, and transcriptomic analysis suggested oxidative stress response as a key target mechanism of this function. Oxidative stress is a hallmark of fetal alcohol toxicity, and we found increased penetrance of facial and ocular malformations in alx1 mutants exposed to ethanol, consistent with a protective role for alx1 against ethanol toxicity.
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Project description:Cell atlas of the human ocular anterior segment: Tissue-specific and shared cell types
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Project description:FOXE3 is a lens specific transcription factor that has been associated with anterior segment ocular dysgenesis. To determine the transcriptional target(s) of FOXE3 that are indispensable for the anterior segment development, we examined the transcriptome and the proteome of cells expressing truncated FOXE3 responsible for Peters anomaly identified through linkage-coupled next-generation whole exome sequencing. We found that DNAJB1, an autophagy-associated protein, was the only candidate exhibiting differential expression in both screens. We confirmed the candidacy of DNAJB1 through chromatin immunoprecipitation and luciferase assays while knockdown of DNAJB1 in human lens epithelial cells resulted in mitotic arrest. Subsequently, we targeted dnajb1a in zebrafish through injection of a splice-blocking morpholino. The dnajb1a morphants exhibited underdeveloped cataractous lenses with persistent apoptotic nuclei. In conclusion, we have identified DNAJB1 as a transcriptional target of FOXE3 in a novel pathway that is crucial for development of the anterior segment of the eye. Human Embryonic Kidney (HEK293FT) cells were transfected with the expression vector (pT-RexTM-DEST30) harboring either the wild type or the mutant (C240*) FOXE3 ORF (open reading frame). The experimental design included a total of eight biological replicates of cells expressing the wild type and eight replicates of mutant FOXE3 along with eight non-transfected controls. Cells were harvested 24-hour post-transfection and subjected to total RNA isolation for the preparation of whole transcriptome next-generation sequencing libraries. Initially, we examined the quality of transcriptome libraries on a MiSeq genome analyzer. Subsequent to confirmation of the quality, all libraries were paired-end sequenced (2 x 100 bp) using Illumina TruSeq Cluster V3 flow cell at a concentration of 13.0 pM in two separate lanes (12 bar-coded mRNA pooled libraries in each lane) on a HiSeq 2000 genome analyzer.
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